Abstract
Objective
To analyze the expression of hypoxia-inducible factor-1α (HIF-1α) and survivin in breast cancer, and different molecular subtypes of breast cancer and to assess their relationship with recurrence and prognosis.
Methods
The expression levels of HIF-1α and survivin genes in breast cancer were investigated using bioinformatics. Their protein expression levels were then verified through immunohistochemistry (IHC), and their relationship with recurrence and prognosis was assessed.
Results
Expression levels of HIF-1α and survivin genes and proteins were increased in breast cancer tissues compared with normal tissues. Both were associated with clinical features of breast cancer and differentially expressed in different molecular subtypes of breast cancer, and both are related to the signal pathway of breast cancer growth and invasion. HIF-1α and survivin gene and protein expression levels were correlated, and both were associated with breast cancer recurrence (R = 0.380, P < 0.05; R = 0.673, P < 0.05, respectively). According to The Cancer Genome Atlas (TCGA) database, HIF1A and BIRC5 gene were not associated with breast cancer prognosis (P ≥ 0.05); however, HIF-1α and survivin protein were associated with recurrence patient's overall survival (OS) (P < 0.05).
Conclusion
HIF-1α and survivin are highly expressed in breast cancer and can be used as potential biomarkers to predict recurrence and assess prognosis.
Keywords: Breast cancer, HIF-1α, Survivin, Recurrence, Prognosis
Graphical abstract
1. Introduction
In recent years, the incidence of breast cancer has been increasing, seriously threatening women's health and quality of life [1]. Individualized treatment based on surgical resection accompanied by radiotherapy is available [2]; however, recurrence occurs in approximately 20% of patients after initial treatment [3]. This recurrence is often accompanied by a poor prognosis. Therefore, identifying markers that predict recurrence or poor prognosis is important for patients with breast cancer.
Hypoxia-inducible factor-1α (HIF-1α) is a G protein-coupled receptor whose overexpression has been identified in various cancers. It activates tumor cells to generate blood vessels that supply oxygen and nutrients, promoting cell proliferation, invasion, and metastasis [4,5]. Survivin is an evolutionarily conserved eukaryotic protein that not only significantly inhibits the apoptosis of tumor cells but also induces cell cycle arrest and promotes tumor survival [6,7]; both these actions should be considered as potential tumor-related therapeutic targets.
Previous studies have suggested that HIF-1α promotes oncogenic phenotypes such as cell viability, proliferation, invasion and migration, and that survivin has unique and distinct functions in cell cycle regulation and apoptotic pathways and is associated with poor prognosis, but it remains unknown how they are expressed in breast cancer patients and whether either plays a role in recurrence in breast cancer patients. In this study, our group conducted a pan-cancer analysis and explored the expression of genes for HIF-1α and survivin; also known as HIF1A and BIRC5 in breast cancer and different molecular subtypes [8] to assess their value in predicting recurrence and prognosis in patients with breast cancer.
2. Samples
For this study, we selected 100 patients with breast cancer treated at the Affiliated Cancer Hospital of Xinjiang Medical University (XJMU) range from 2006 to 2021. Stratified random sampling was used to divide the patients into two groups, one for patients with chest wall recurrence and one for patients with no recurrence, with 50 patients in each group. Inclusion criteria were: 1) All patients underwent lumpectomy; 2) patients' pathological diagnosis had to be breast cancer; 3) patients were treated with a combination therapy for more than 3 months; 4) all selected patients agreed to provide their data or tissue samples in this study. Exclusion criteria were: 1) patients who are unwilling to provide samples or participate in this study; 2) patients with other diseases that affect the quality of life (such as metabolic diseases, reproductive system tumors, immune system diseases, mental diseases) 3) patients with diseases related to hormone levels (such as patients with endometrial cancer, or cancer during pregnancy and lactation); 4) patients who have received immunotherapy, targeted therapy or other clinical trials in the past three months. The study protocol was approved by the Ethics Committee of the Affiliated Cancer Hospital of XJMU and we got informed consent was obtained from all the patients.
3. Methods
3.1. Detection of gene expression
The expression levels of HIF1A and BIRC5 in normal and tumor tissues were investigated through The TCGA database, and P < 0.05 was considered a statistical difference. TISIDB is a web portal for tumor and immune system interaction, which integrates multiple heterogeneous data types [9]. We used TISIDB to study the expression of HIF1A and BIRC5 in different subtypes of breast cancer. UALCAN provides charts and graphs describing protein-coding expression profiles and patient survival information [10]. This study investigated the expression of HIF1A and BIRC5 in different molecular subtypes of breast cancer and their correlation with clinical features and survival prognosis.
3.2. Detection of protein expression
This study investigated HIF-1α and survivin expression based on the Human Protein Atlas (HPA) [11], a large database for the differential expression of proteins in normal and tumor tissues. The excised tissues from these 100 patients were made into wax-block samples, each containing both cancerous and normal tissues, for subsequent IHC experiments. We used IHC to detect the distribution and localization of HIF-1α and survivin, and the results were evaluated independently by two pathologists. A semi-quantitative scoring system [12] was used to assess the intensity of staining (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and the percentage of positive cells to cells with similar scores. Positive cells were counted in 5 randomly selected fields of view in a 200× field of view. The percentage of positive cells was measured for each image and the average number of stained cells was determined. (No positive cells were counted as 0, 0–20% as 1, 21–50% as 2, 51–80% as 3 and 80%–100% as 4). The staining intensity of the cells and the percentage of positivity were multiplied together to give an Immunological Activity Score (I.A.) for each case, with 0–1 being "-", 2–4 being "+", 5–7 being "++" and 8 or more being "+++". In this study, "- to +" was defined as low expression and "++ to ++++" as high expression.
3.3. Co-expression and correlation analysis of HIF1A and BIRC5
Our group performed gene ontology (GO) analyses using HIF1A and BIRC5. GO is the international standard classification system for gene function after screening specific genes according to the experimental purpose [13]. We explored the genes associated with HIF1A and BIRC5 expression levels in cancer and the correlation between the two.
3.4. Survival analysis
Gene expression was performed in line with the aforementioned databases. R language was used for statistical analysis, and the ggplot2 package was used for visualization. A Wilcoxon rank-sum test was used to assess HIF1A and BIRC5 expression levels in unpaired and paired tissues, respectively. Logistic regression was used to assess the correlation between clinical characteristics and HIF1A and BIRC5 expression levels. All tests were two-sided, and P < 0.05 was considered statistically significant. Chi-squared test was used to investigate the relationship between HIF-1α and survivin expression and clinicopathological parameters. Spearman's correlation was used to analyze the association between molecules, and Kaplan–Meier tests was used for survival analysis, with P < 0.05 considered statistically significant.
4. Results
4.1. Differential expression of HIF1A and BIRC5 genes
The study analyzed 112 sets of paired data from 18 types of normal human tissues and tissues from tumors included in the TCGA database. The results detected significant differences in the expression levels of HIF1A and BIRC5 in normal and cancerous tissues (Fig. 1) We found significant differences between HIF1A and BIRC5 in breast cancer tissue and normal tissue (P < 0.05), as detailed in Table 1.
Fig. 1.
Differential expression of HIF1A and BIRC5 in normal tissues and cancer tissues. a HIF1A expression in normal tissues and cancer tissues. b BIRC5 expression in normal tissues and cancer tissues. Notes:* is P < 0.05, ** is P < 0.01, and *** is P < 0.001. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinomaand endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colonadenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.
Table 1.
HIF1A and BIRC5 expression in normal and tumor tissues in TCGA database.
| N | Median | Mean | SD | Statistics | P | |
|---|---|---|---|---|---|---|
| HIF1A | 2.765 | 0.007 | ||||
| Normal tissues | 112 | 6.127 | 6.048 | 0.629 | ||
| Turmor tissues | 112 | 6.456 | 6.314 | 0.949 | ||
| BIRC5 | 18.759 | 0.000 | ||||
| Normal tissues | 112 | 1.584 | 4.755 | 1.369 | ||
| Turmor tissues | 112 | 4.927 | 4.755 | 1.369 |
Note: The bold part indicates P < 0.05.
We investigated the relationship between HIF1A and BIRC5 with clinical features of breast cancer. The expression levels of HIF1A were associated with the estrogen and progesterone (ER/PR) status, human epidermal growth factor receptor 2 (HER2) status, and tumor pathological type of patients with breast cancer (P < 0.05). The expression of BIRC5 was related to the age, T stage, pathological stage, tumor pathological type, ER/PR status, and HER2 status of patients with breast cancer (P < 0.05), as detailed in Table 2.
Table 2.
Relationship between HIF1A and BIRC5 with clinical features of BRCA in TISIDB database.
| Clinical features | HIF1A |
BIRC5 |
||||
|---|---|---|---|---|---|---|
| Low expression | High expression | P | Low expression | High expression | P | |
| N | 541 | 542 | 541 | 542 | ||
| T-Stage | 0.199 | 0.001 | ||||
| T1 | 141 (13.1%) | 136 (12.6%) | 176 (16.3%) | 101 (9.4%) | ||
| T2 | 305 (28.2%) | 324 (30%) | 276 (25.6%) | 353 (32.7%) | ||
| T3 | 79 (7.3%) | 60 (5.6%) | 73 (6.8%) | 66 (6.1%) | ||
| T4 | 14 (1.3%) | 21 (1.9%) | 15 (1.4%) | 20 (1.9%) | ||
| N-Stage | 0.481 | 0.105 | ||||
| N0 | 250 (23.5%) | 264 (24.8%) | 255 (24%) | 259 (24.3%) | ||
| N1 | 188 (17.7%) | 170 (16%) | 182 (17.1%) | 176 (16.5%) | ||
| N2 | 52 (4.9%) | 64 (6%) | 48 (4.5%) | 68 (6.4%) | ||
| N3 | 38 (3.6%) | 38 (3.6%) | 45 (4.2%) | 31 (2.9%) | ||
| Metastasis | 0.630 | 0.609 | ||||
| M0 | 424 (46%) | 478 (51.8%) | 436 (47.3%) | 466 (50.5%) | ||
| M1 | 11 (1.2%) | 9 (1%) | 8 (0.9%) | 2 (1.3%) | ||
| TNM Stage | 0.776 | 0.003 | ||||
| I | 93 (8.8%) | 88 (8.3%) | 112 (10.6%) | 69 (6.5%) | ||
| II | 308 (29.1%) | 311 (29.3%) | 286 (27%) | 333 (31.4%) | ||
| Ⅲ | 119 (11.2%) | 123 (11.6%) | 122 (11.5%) | 120 (11.3%) | ||
| IV | 11 (1%) | 7 (0.7%) | 8 (0.8%) | 10 (0.9%) | ||
| Pathological type | 0.001 | 0.001 | ||||
| Infiltrating ductal carcinoma | 355 (36.3%) | 417 (42.7%) | 318 (32.5%) | 454 (46.5%) | ||
| Invasive lobular carcinoma | 126 (12.9%) | 79 (8.1%) | 161 (16.5%) | 44 (4.5%) | ||
| Age(y) | 0.286 | 0.002 | ||||
| ≤60 | 291 (26.9%) | 310 (28.6%) | 274 (25.3%) | 327 (30.2%) | ||
| >60 | 250 (23.1%) | 232 (21.4%) | 267 (24.7%) | 215 (19.9%) | ||
| PR status | 0.001 | 0.001 | ||||
| Negative | 124 (12%) | 218 (21.1%) | 97 (9.4%) | 245 (23.7%) | ||
| Positive | 392 (37.9%) | 296 (28.6%) | 420 (40.6%) | 268 (25.9%) | ||
| Uncertain | 3 (0.3%) | 1 (0.1%) | 2 (0.2%) | 2 (0.2%) | ||
| ER status | 0.001 | 0.001 | ||||
| Negative | 72 (7%) | 168 (16.2%) | 51 (4.9%) | 189 (18.3%) | ||
| Positive | 448 (43.3%) | 345 (33.3%) | 468 (45.2%) | 325 (31.4%) | ||
| Uncertain | 0 (0%) | 2 (0.2%) | 0 (0%) | 2 (0.2%) | ||
| HER2 status | 0.005 | 0.047 | ||||
| Negative | 275 (37.8%) | 283 (38.9%) | 290 (39.9%) | 268 (36.9%) | ||
| Positive | 59 (8.1%) | 98 (13.5%) | 66 (9.1%) | 91 (12.5%) | ||
| Menstrual status | 0.219 | 0.138 | ||||
| Premenopausal | 120 (12.3%) | 109 (11.2%) | 105 (10.8%) | 124 (12.8%) | ||
| Postmenopausal | 350 (36%) | 353 (36.3%) | 368 (37.9%) | 335 (34.5%) | ||
| Uncertain | 15 (1.5%) | 25 (2.6%) | 17 (1.7%) | 23 (2.4%) | ||
| Radiation therapy | 0.755 | 0.541 | ||||
| Not | 220 (22.3%) | 214 (21.7%) | 226 (22.9%) | 208 (21.1%) | ||
| Yes | 287 (29.1%) | 266 (27%) | 276 (28%) | 277 (28.1%) | ||
Note: The bold part indicates P < 0.05.
The TISIDB database is a tumor immunoassay database that shows the expression levels of different molecular subtypes in different cancers and in one cancer separately. We investigated the relationship between HIF1A and BIRC5 expression levels and the molecular typing of breast cancer in the TISIDB database. Breast cancer is a highly heterogeneous tumor and is currently classified as Luminal, HER2-overexpressing and triple-negative breast cancer (TNBC), our results suggested that HIF1A expression levels were higher in patients with HER2-overexpressing (P < 0.05) and BIRC5 expression levels were higher in patients with TNBC(P < 0.05). See Fig. 2a–b. As TNBC can be further divided into six subtypes: luminal androgen receptor (LAR), basaloid-1 (BL-1), basaloid-2 (BL-2), immunomodulatory (IM), mesenchymal (M) and mesenchymal stem cell (MSC). The Ualcan database, which was selected to further analyze the expression of both in the molecular subtypes of triple-negative breast cancer, suggesting that the expression of HIF1A was higher in the luminal androgen receptor subtype than in the other subtypes (P < 0.05). See Fig. 2c–d.
Fig. 2.
Expression of HIF1A and BIRC5 in breast cancer. a BIRC5 expression with breast cancer subtype in the TISIDB database. 2b HIF1A expression with breast cancer subtype in the TISIDB database. 2c HIF1A expression with and molecular types of breast cancer in the UALCAN database. 2d BIRC5 expression with molecular types of breast cancer in the UALCAN database. Note: 2a,b: Vertical coordinate indicates that the corrected P value is taken as −log2, and the horizontal coordinate indicates the molecular typing of the breast cancer. 2c,d: Vertical coordinates indicate corrected P values taken as −log10, and the horizontal coordinates indicate the various molecular subtypes of breast cancer.
4.2. Differential expression of HIF-1α and survivin proteins
In this study, HIF-1α and survivin expression levels were analyzed using the HPA database, which covers almost all normal and human tumor tissues. The results suggest that HIF-1α is expressed in the nucleus and partially in cytoplasm, is highly expressed in normal human bone marrow, and is expressed in a variety of malignant tumors such as those of breast cancer, lung cancer, glioblastoma, and gastric cancer. Survivin is expressed in cytoplasm and is highly expressed in bone marrow, lymphoid tissue, and testis; differences were detected in single-cell type-specific expression, with a high level of expression in spermatocytes, erythrocytes, and cytotrophoblasts. Survivin is expressed in many types of human tumors, whereas no expression or only low levels are present in the corresponding normal tissues and cells. See Fig. 3a–b.
Fig. 3.
Expression of HIF-1α and survivin in HPA database and 100 patients. 3a Expression of HIF-1α in HPA. 3b Expression of survivin in HPA. 3c Expression of HIF-1α in normal tissue of 100 patients. 3d Expression of survivin in normal tissue of 100 patients. 3e Expression of HIF-1α in tumor tissue of 100 patients. 3f Expression of survivin in tumor tissue of 100 patients. Note: 3a,b. Horizontal coordinates indicate the normal body tissues of each system. Normal tissues include brain tissue (cerebral cortex, cerebellum, hippocampus), endocrine system (thyroid, parathyroid, adrenal), respiratory system (nasopharynx, bronchi, lungs), digestive system (oral mucosa, salivary glands, esophagus, stomach, duodenum, small intestine, colon, rectum, liver, gallbladder, pancreas), urinary system (kidneys, bladder), male and female reproductive system (testes, epididymis, seminal vesicles, prostate, vagina, ovaries, fallopian tubes, endometrium, cervix, placenta, breast), skeletal-muscular system (cardiac muscle, smooth muscle, skeletal muscle), soft tissue, adipose tissue, skin, immune system (spleen, lymph nodes, tonsils, bone marrow), and tumor tissue, including tumor tissue corresponding to the above normal tissues.3c-f.Expression of HIF-1α and survivin in 100 patients by IHC.Observed under a microscope at 200× magnification.
We focused on the expression of HIF-1α and survivin in our selected patient tissues using IHC. See Fig. 3c–f. In the stained sections of normal and tumor tissues from 100 patients, the positive rate of HIF-1α was 51% in tumor tissues and 5% in normal tissues (χ2 = 52.48, P < 0.05). The positive rate of survivin was 68% and 10% in tumor and normal tissues, respectively (χ2 = 72.83, P < 0.05). The expression level of HIF-1α was 70% in tumor tissues from patients who had relapsed and 32% in tumor tissues from patients without recurrence (χ2 = 14.45, P < 0.05). The expression level of survivin was 86% in tumor tissues from patients who had relapsed and 50% in tumor tissues from patients with no recurrence (χ2 = 13.51, P < 0.05). The expression levels of HIF-1α and survivin were higher in the tumors of patients with recurrence than in those of patients with nonrecurrence.
A univariate analysis suggested that the expression levels of HIF-1α and survivin were associated with pathological stage, ER/PR status, HER2 expression, and radiotherapy (P < 0.05). HIF-1α and survivin levels were not associated with age, menstrual status, pathological type, or therapies such as chemotherapy, endocrine therapy, or targeted therapy (P ≥ 0.05). High levels of HIF-1α and survivin expression were positively correlated with recurrence (R = 0.38, P < 0.05; R = 0.67, P < 0.05, respectively), as detailed in Table 3.
Table 3.
HIF-1α and Survivin expression in 100 BRCA patients with different characteristics.
| Clinical characteristics | N | HIF-1α |
Survivin |
||||
|---|---|---|---|---|---|---|---|
| High expression | Low expression | P | High expression | Low expression | P | ||
| N | 100 | 51 | 49 | 69 | 31 | ||
| Age(y) | 0.619 | 0.572 | |||||
| <50 | 67 | 33 | 34 | 45 | 22 | ||
| ≥50 | 33 | 18 | 15 | 24 | 9 | ||
| Menstrual status | 0.315 | 0.422 | |||||
| Premenopausal | 70 | 38 | 32 | 50 | 20 | ||
| Postmenopausal | 30 | 13 | 17 | 19 | 11 | ||
| T-Stage | 0.006 | 0.047 | |||||
| T1+T2 | 77 | 45 | 32 | 52 | 25 | ||
| T3+T4 | 23 | 6 | 17 | 12 | 11 | ||
| N-Stage | 0.000 | 0.049 | |||||
| N0 | 25 | 17 | 8 | 19 | 6 | ||
| N1 | 27 | 20 | 7 | 23 | 4 | ||
| N2 | 23 | 8 | 15 | 12 | 11 | ||
| N3 | 25 | 6 | 19 | 15 | 10 | ||
| TNM Stage | 0.000 | 0.020 | |||||
| I + II | 43 | 31 | 12 | 35 | 8 | ||
| Ⅲ+IV | 57 | 20 | 37 | 34 | 23 | ||
| Hormone Receptor Status | 0.814 | 0.386 | |||||
| ER or PR Positive | 42 | 22 | 20 | 27 | 15 | ||
| ER and PR Negative | 58 | 29 | 29 | 42 | 16 | ||
| Her-2 Status | 0.000 | 0.000 | |||||
| Negative | 41 | 13 | 28 | 20 | 21 | ||
| Positive | 59 | 39 | 20 | 49 | 10 | ||
| Molecular subtype | 0.000 | 0.036 | |||||
| LuminalA | 13 | 1 | 12 | 6 | 7 | ||
| LuminalB | 30 | 22 | 8 | 22 | 8 | ||
| HER2 | 29 | 17 | 12 | 17 | 12 | ||
| TNBC | 28 | 11 | 17 | 24 | 4 | ||
| Pathological type | 0.918 | 0.820 | |||||
| Ductal carcinoma | 8 | 4 | 4 | 5 | 3 | ||
| Infiltrating ductal carcinoma | 87 | 44 | 43 | 61 | 26 | ||
| Uncertain | 5 | 3 | 2 | 3 | 2 | ||
| Radiation therapy | 0.564 | 0.061 | |||||
| Yes | 75 | 37 | 38 | 48 | 27 | ||
| Not | 25 | 14 | 11 | 21 | 4 | ||
| Chemotherapy | 0.722 | 0.082 | |||||
| Yes | 76 | 38 | 38 | 49 | 27 | ||
| Not | 24 | 13 | 11 | 20 | 4 | ||
| Endocrine therapy | 0.070 | 0.213 | |||||
| Yes | 52 | 22 | 30 | 33 | 19 | ||
| Not | 48 | 29 | 19 | 36 | 12 | ||
| Anti-Her-2 therapy | 0.299 | 0.170 | |||||
| Yes | 25 | 15 | 10 | 20 | 5 | ||
| Not | 75 | 36 | 39 | 49 | 26 | ||
| Recurrence | 0.000 | 0.000 | |||||
| Yes | 50 | 35 | 15 | 43 | 7 | ||
| Not | 50 | 16 | 34 | 26 | 24 | ||
Note: The bold part indicates P < 0.05.
We used Cox regression models to analyze the relationship between HIF-1α and survivin and clinical characteristics in the 100 patients with breast cancer, revealing that HIF-1α expression levels are associated with the HER2 subtype of breast cancer (χ2 = 4.494, P = 0.034), whereas expression levels of survivin are associated with the TNBC subtype of breast cancer (χ2 = 6.898, P = 0.009), as detailed in Table 4.
Table 4.
Cox regression of HIF-1α and Survivin expression in different subtypes of patients.
| Molecular subtype | HIF-1α |
Survivin |
||||||
|---|---|---|---|---|---|---|---|---|
| HR | 95%CI | χ2 | P | HR | 95%CI | χ2 | P | |
| Luminal subtype | 0.970 | 0.367–0.255 | 0.004 | 0.951 | 1.021 | 0.395–2.636 | 0.002 | 0.966 |
| HER2 subtype | 2.104 | 1.058–4.184 | 4.494 | 0.034 | 1.509 | 0.851–2.673 | 1.985 | 0.159 |
| TNBC subtype | 0.990 | 0.600–1.634 | 0.002 | 0.969 | 3.683 | 1.392–9.745 | 6.898 | 0.009 |
Note: The bold part indicates P < 0.05.
We observed HIF-1α and survivin expression levels in tumor tissues of patients who had relapsed and those who had not, and the results suggested that in patients who had relapsed, HIF-1α and survivin expression levels were associated with pathological stage, ER and/or PR expression, HER2 expression level, and radiotherapy (P < 0.05) but not with age, menstrual status, pathological type, or treatments such as chemotherapy, endocrine therapy, or targeted therapy (P ≥ 0.05), as detailed in Table 5.
Table 5.
Expression of HIF-1α and Survivin in 50 relapsed patients with clinicopathological features.
| Relapsed patients | N | HIF-1α |
Survivin |
||||
|---|---|---|---|---|---|---|---|
| High expression | Low expression | P | High expression | Low expression | P | ||
| 50 | 35 | 15 | 43 | 7 | |||
| Age(y) | 34 | 21 | 13 | 0.693 | 30 | 4 | 0.507 |
| < 50 | 16 | 14 | 2 | 13 | 3 | ||
| ≥ 50 | |||||||
| Menstrual status | 33 | 30 | 3 | 0.765 | 28 | 5 | 0.744 |
| Premenopausal | 17 | 15 | 2 | 15 | 2 | ||
| Postmenopausal | |||||||
| T-Stage | 32 | 20 | 12 | 0.014 | 25 | 7 | 0.033 |
| T1+T2 | 18 | 17 | 1 | 18 | 0 | ||
| T3+T4 | 50 | 35 | 15 | 43 | 7 | ||
| N-Stage | 0.004 | 0.001 | |||||
| N0 | 8 | 4 | 4 | 4 | 4 | ||
| N1 | 8 | 4 | 4 | 5 | 3 | ||
| N2 | 15 | 15 | 0 | 15 | 0 | ||
| N3 | 19 | 17 | 2 | 19 | 0 | ||
| TNM Stage | 0.001 | 0.003 | |||||
| I + II | 12 | 2 | 10 | 7 | 5 | ||
| Ⅲ+IV | 38 | 33 | 5 | 36 | 2 | ||
| Hormone Receptor Status | 0.004 | 0.013 | |||||
| ER or PR Positive | 21 | 10 | 11 | 15 | 6 | ||
| ER and PR Negative | 29 | 25 | 4 | 28 | 1 | ||
| Her-2 Status | 0.001 | 0.006 | |||||
| Negative | 10 | 2 | 8 | 5 | 5 | ||
| Positive | 40 | 33 | 7 | 38 | 2 | ||
| Molecular subtype | 0.004 | 15 | 6 | 0.013 | |||
| LuminalA | 21 | 10 | 11 | 28 | 1 | ||
| LuminalB | 29 | 25 | 4 | 5 | 5 | ||
| HER2 | 10 | 2 | 8 | 38 | 2 | ||
| TNBC | 40 | 33 | 7 | 2 | 2 | ||
| Pathological type | 0.214 | 0.082 | |||||
| Ductal carcinoma | 4 | 2 | 2 | 38 | 5 | ||
| Infiltrating ductal carcinoma | 43 | 32 | 11 | 3 | 0 | ||
| Uncertain | 3 | 1 | 2 | ||||
| Radiation therapy | 0.030 | 40 | 2 | 0.001 | |||
| Yes | 42 | 32 | 10 | 3 | 5 | ||
| Not | 8 | 3 | 5 | ||||
| Chemotherapy | 0.773 | 0.069 | |||||
| Yes | 38 | 27 | 11 | 31 | 7 | ||
| Not | 12 | 8 | 4 | 12 | 0 | ||
| Endocrine therapy | 0.073 | 0.069 | |||||
| Yes | 23 | 19 | 4 | 22 | 1 | ||
| Not | 27 | 16 | 11 | 21 | 6 | ||
| Anti-Her-2 therapy | 0.527 | 0.446 | |||||
| Yes | 13 | 10 | 3 | 12 | 1 | ||
| Not | 37 | 25 | 12 | 31 | 6 | ||
Note: The bold part indicates P < 0.05.
4.3. Co-expression and correlation analysis of HIF1A and BIRC5
This study used a gene co-expression network to obtain genes interacting with HIF1A and BIRC5. These results suggest that HIF1A is a major transcriptional regulator of the adaptive response to hypoxia, playing a crucial role in promoting tumor angiogenesis. Major biological processes (BP) include mitochondrial autoregulation, actin cytoskeleton organization, angiogenesis, the positive regulation of cytokine production, and an epithelial to mesenchymal transition. Cellular components (CC) involve transcription factor complexes, axonal fraction, and nuclear transcription factor complexes. Molecular functions (MF) include the positive regulation of transcription through RNA polymerase II. In terms of the 981 genes associated with BIRC5, BP are microtubule cytoskeleton organization, sister chromatid separation, spindle organization, mitotic nuclear division, and the negative regulation of peptidase activity, endopeptidase activity, and protein processing. CC are an enriched chromosomal mitogenic region, condensed chromosome motor region, spindles, and microtubules. MF are involved in ubiquitin protein transferase activity, enzyme inhibitor activity, cysteine-type endopeptidase inhibitor activity, and microtubule binding. See Fig. 4a–b.
Fig. 4.
GO processes and relationship between HIF-1α and survivin at the gene and protein levels. 4a GO processes of HIF1A. 4b GO processes of BIRC5. 4c Relationship between HIF1A and BIRC5 at the gene levels. 4d Relationship between HIF-1α and Survivin at the gene levels. Note: 4a,b: Red squares on the left are HIF1A and BIRC5 genes, blue circles in the middle are all genes co-expressed with HIF1A and BIRC5, green on the right are co-expressed genes enriched biologically by GO.The size of the bubble indicates the number of genes enriched. The color of the bubble indicates the significance of the P value: the redder the bubble, the smaller the P value and the more significant the result, and the bluer the bubble, the larger the P value.4c,d: Relationship between HIF-1α and survivin at the gene and protein levels.
We found that the expression of HIF1A and BIRC5 were correlated at the gene and protein levels (R = 0.133, P < 0.05, R=0.62,P < 0.05). See Fig. 4c–d.
4.4. Prognosis analysis
This study analyzed the association of HIF1A and BIRC5 with the prognosis of patients with breast cancer according to the TCGA database. The results demonstrated that HIF1A and BIRC5 were not significantly associated with the OSrate of patients with breast cancer (hazard ratio (HR) = 0.68, P = 0.11; HR = 1.53, P = 0.05, respectively), See Fig. 5a–b. Of the 100 patients with breast cancer included in this study, HIF-1α and survivin proteins were not associated with OS (χ2 = 1.373, P = 0.241; χ2 = 0.034, P = 0.853, respectively), with the results similar to those of genes in the database, See Fig. 5c–d. However, in the 50 patients who had relapsed, HIF-1α and survivin were associated with OS (χ2 = 11.104, P = 0.001; χ2 = 6.097, P = 0.014, respectively), See Fig. 5e–f.
Fig. 5.
Association between HIF-1α and survivin gene and protein expression levels and prognosis. 5a The association between HIF1A expression in the TCGA database and OS in breast cancer patients. 5b The association between BIRC5 expression in the TCGA database and OS in breast cancer patients. 5c The association between HIF-1α protein expression levels and prognosis in 100 breast cancer patients. 5d The association between survivin protein expression levels and prognosis in 100 breast cancer patients. 5e The relationship between HIF-1α protein expression levels and prognosis in 50 patients with recurrent breast cancer. 5f The relationship between survivin protein expression levels and prognosis in 50 patients with recurrent breast cancer.
5. Discussion
Breast cancer is the leading cause of morbidity and mortality in women for all types of malignancies [14]. In recent decades, great progress has been made in the management of breast cancer, but a high percentage of patients with breast cancer still experience recurrence after tumor removal [15]. Therefore, it is important to identify markers that predict recurrence or poor prognosis and to intervene with therapeutic measures in breast cancer patients [16]. HIF-1α is overexpressed in various cancers and that its action activates tumor cell angiogenesis, supplying oxygen and nutrients to promote their growth and proliferation, invasion, and metastasis [17]. Survivin is expressed in a variety of tumor cells, which can promote the apoptosis of tumor cells and has a strong function in apoptosis-related signal pathways [18]. Few studies have linked these two indicators; therefore, this study examined how they are related in breast cancer.
This study identified a significant difference in the expression levels of HIF1A and BIRC5 in normal and cancerous tissues. As breast cancer is a highly heterogeneous disease and there is a lack of screening tools for specific types of breast cancer, we used a bioinformatics tool - ULACAN - and found that HIF1A and BIRC5 were differentially expressed in different molecular subtypes of breast cancer. Our results revealed that in TNBC subtypes, HIF1A expression levels are higher in the LAR subtype than in other subtypes. This may be because HIF1A is associated with the cellular recycling pathway, and the overall function of this pathway is synergistic through intracellular transport [19]. The poor prognosis of patients with intraluminal androgen receptors is closely related to the androgen receptor signaling pathway, and the inhibition of HIF1A may sensitize patients to androgen inhibitors, which would help improve their prognosis. We found that BIRC5 expression is higher in the BL-2 subtype than in other subtypes. The BL-2 subtype is mainly detected in young patients and is characterized by a low tumor grade and low invasive capacity of breast cancer, commonly found in invasive lobular and medullary carcinomas [20] However, these studies have not been mentioned in previous reports, and future trials of novel targeted therapies should be conducted in clinical settings based on the mutation types of different TNBC subtypes.
The HPA database revealed that HIF-1α and survivin were not expressed or were only present at low levels in normal human tissues but were highly expressed in their corresponding tumor tissues. We performed IHC experiments to validate the expression of HIF-1α and survivin in real-world patients and the results indicating that HIF-1α and survivin are highly expressed in patients who had relapsed than in those who had not. Studies have demonstrated that HIF-1α is associated with tumor recurrence in tumors such as those related to pancreatic cancer [21] and nasopharyngeal angiofibroma [22]. Additional studies confirm Survivin's association with high tumor grade cancers and recurrence [23]. The present study identified that HIF-1α and survivin were associated with tumor recurrence, which is consistent with ours Recently, Collin conducted two studies on breast cancer in Danish patients and identified that HIF-1α expression in ER-negative tumors was associated with early recurrence in female patients with breast cancer [24]; however, survivin expression was not associated with breast cancer recurrence, but the ratio of survivin in the cytoplasm to the nucleus may be associated with recurrence [25]. The results of the present study differ from those of the Danish study. This may be because the Danish study included more patients with positive ER expression who were treated with tamoxifen, which are key factors affecting the recurrence of breast cancer. In addition, the availability of targeted therapies and radiation therapy is an important factor in the recurrence of breast cancer; these factors were not mentioned in the Danish study. Our study encompassed the impact of clinical characteristics and treatment on breast cancer recurrence and prognosis. To our knowledge, this study is the first to combine HIF-1α and survivin in a breast cancer-related study.
The present study demonstrated that HIF1A and BIRC5 are positively correlated in gene and protein. GO analysis revealed that HIF1A and BIRC5 are involved in pathways associated with carcinogenesis. These signaling pathways cause activation of the intracellular segment of the cell surface receptor, which transduces extracellular signals into the cell [26,27]. Therefore, the use of inhibitors of the HIF1A and BIRC5 pathways may be considered in the future to control tumor growth. Peng et al. [28] showed that HIF1α can directly bind to the survivin promoter and downregulate its gene expression pattern, and this gene regulation mechanism is further supported by the study of Zhang et al. [29], who validated the association of HIF1α with survivin at the gene level. Our group's experiments also confirmed the association at the protein level. Therefore, when considering treatment strategies for patients, one may choose to control tumor recurrence by suppressing the expression of these proteins to reduce the cost of treatment, rather than considering strategies that suppress the genes.
This study investigated the relationship between HIF-1α and Survivin and the clinical characteristics of patients. We found that the average time from initial diagnosis to the presentation of recurrence in patients with breast cancer was 2.24 years, which is different from the results of other studies [30]. This may be related to the bias of case selection, number of patients, and choice of systemic treatment. Our study identified the average time at which patients with breast cancer are most likely to experience recurrence, which may encourage patients to follow their doctor's instructions more closely and provide a proactive strategy for early treatment. In the 50 relapsed patients, high levels of HIF-1α and survivin had shorter OS of patiens than those with low levels. In a recent meta-analysis involving 2933 patients, researchers found that high levels of HIF-1α expression were associated with poor OS rates (HR = 1.46, 95%CI: 1.12–1.92, P < 0.05) [31]. Another meta-analysis pooling 3259 patients with breast cancer revealed a positive association between poor prognosis (HR = 1.37, 95%CI: 1.12–1.68, P < 0.05) and survivin expression [32]. The data from these studies were based on medical record reviews, thus reducing the possibility of a misclassification of the results. Our results are consistent with those of these meta-analyses, indicating that an increased expression of HIF-1α and survivin is associated with a poorer prognosis in breast cancer. According to these results, it can be concluded that risk score models for HIF-1α and survivin can serve as a powerful prognostic biomarker.
The study has some shortcomings. First, it is very simple to use the TCGA database to study interested genes for the survival of patients, but there are still many researchers who do not use the TCGA database. Therefore, in order to make the research results more convincing, tissue samples from more patients can be included in future studies. Second, the samples we selected were postoperative specimens, some of which had been stored for a long time. Therefore, follow-up studies should extract fresh tumor tissues to study HIF-1α and survivin protein expression. In addition, blood samples of patients are also worth collecting. It has been suggested that HIF-1α and survivin are differentially expressed in blood and tissues, so it is possible to systematically study the expression levels of target genes or proteins in different samples in the future.
6. Conclusion
HIF-1α and survivin genes and proteins are differentially expressed in normal and tumor tissues; their expression levels correlate with the clinical features of breast cancer, and they are differentially expressed in different molecular subtypes of breast cancer. The expression levels of HIF-1α and survivin are associated with patient recurrence and survival, and there is a need to develop combined HIF-1α and survivin inhibitors in the future to provide effective treatment strategies for patients.
Author contribution statement
Qian Cao: Conceived and designed the experiments; Performed the experiments; Wrote the paper.
Munire Mushajiang: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.
Cheng-qiong Tang: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.
Xiuqing Ai: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.
Funding statement
Qian Cao was supported by Natural Science Foundation of Xinjiang Uygur Autonomous Region [2021D01C417].
Data availability statement
Data will be made available on request.
Declaration of interest's statement
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Ethics approval and consent to participate
The study protocol was approved by the Ethics Committee of The Affiliated Cancer Hospital of Xinjiang Medical University (Approval number: K-2021070). All patients were obtained informed consent before the experiment.
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Associated Data
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Data Availability Statement
Data will be made available on request.